This grant proposal represents the third competitive renewal (Years 16-20) of a highly productive research program investigating fundamental cellular and molecular signaling pathways relevant to human neurodegenerative disorders. Over the last 15 years of funding from this grant, our studies have characterized in detail a signaling cascade connecting the oxidative liberation of intracellular zinc to a neuronal cell death-enabling increase in the plasma membrane insertion of Kv2.1 channels. This process mediates a loss of cytoplasmic potassium, a requisite step for optimal protease and nuclease catalytic activity during programmed and other forms of cell death. In the current funding period (Years 11-15), we have studied the phosphorylation events leading to the syntaxin-dependent exocytotic insertion of Kv2.1 into the neuronal plasma membrane following injury. We also investigated the role of Kv2.1 somatodendritic clusters as dominant membrane channel insertion hubs during apoptosis. Most importantly, we established a novel in vivo neuroprotective approach to stroke injury by developing a cell-permeant peptide that interferes with a critical Kv2.1 interaction with syntaxin. Our work strongly indicates that specifically targeting Kv2.1-facilitated cell death processes can provide mechanistically driven, novel therapeutic strategies for neuroprotection. In this application we propose to decisively move forward with this strategic objective by: (i) characterizing the properties of novel neuroprotective peptides and derived small molecule analogs targeting the syntaxin/Kv2.1 interaction; (ii) establishing prototypical neuroprotective tools aimed at dispersing Kv2.1 somatodendritic clusters, and (iii) devising an innovative neuroprotective strategy that transfers a normally silent gene to neurons, designed to express a Kv2.1-targeted modulatory protein when cells are lethally injured. We thus have adopted three unique and potentially transformative strategies, all based on the premise that cell death-inducing pathways require a set of common conditions to operate optimally. The loss of intracellular potassium via a surge of Kv2.1-mediated ionic currents may constitute a widespread, if not ubiquitous, requirement for programmed cell death in many types of neurons. As such, the experiments described here are aimed at translating the cellular and molecular pathways we have characterized with long-term funding from this grant, into rational therapeutic approaches to neurodegeneration.